Published online before print January 22, 2009, doi: 10.1161/CIRCRESAHA.108.188748
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© 2009 American Heart Association, Inc.
Cellular Biology |
TRPC1 Associates With BKCa Channel to Form a Signal Complex in Vascular Smooth Muscle Cells
From the Institute of Vascular Medicine, Li Ka Shing Institute of Health Sciences, and Department of Physiology, Faculty of Medicine, the Chinese University of Hong Kong.
Correspondence to Xiaoqiang Yao, PhD, Department of Physiology, The Chinese University of Hong Kong, Hong Kong, China. E-mail yao2068{at}cuhk.edu.hk
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Abstract |
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TRPC1 (transient receptor potential canonical 1) is a Ca2+-permeable cation channel involved in diverse physiological function. TRPC1 may associate with other proteins to form a signaling complex, which is crucial for channel function. In the present study, we investigated the interaction between TRPC1 and large conductance Ca2+-sensitive K+ channel (BKCa). With the use of potentiometric fluorescence dye DiBAC4(3), we found that store-operated Ca2+ influx resulted in membrane hyperpolarization of vascular smooth muscle cells (VSMCs). The hyperpolarization was inhibited by an anti-TRPC1 blocking antibody T1E3 and 2 BKCa channel blockers, charybdotoxin and iberiotoxin. These data were confirmed by sharp microelectrode measurement of membrane potential in VSMCs of intact arteries. Furthermore, T1E3 treatment markedly enhanced the membrane depolarization and contraction of VSMCs in response to several contractile agonists including phenylephrine, endothelin-1, and U-46619. In coimmunoprecipitation experiments, an antibody against BKCa
-subunit [BKCa()] could pull down TRPC1, and moreover an anti-TRPC1 antibody could reciprocally pull down BKCa(). Double-labeling immunocytochemistry showed that TRPC1 and BKCa were colocalized in the same subcellular regions, mainly on the plasma membrane, in VSMCs. These data suggest that, TRPC1 physically associates with BKCa in VSMCs and that Ca2+ influx through TRPC1 activates BKCa to induce membrane hyperpolarization. The hyperpolarizing effect of TRPC1-BKCa coupling could serve to reduce agonist-induced membrane depolarization, thereby preventing excessive contraction of VSMCs to contractile agonists.
Key Words: TRPC1 • BKCa • physical coupling • hyperpolarization • vascular smooth muscle cells
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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TRPC1 (transient receptor potential canonical 1) is among the most-studied TRP channels.1 The channel is widely expressed in many cell types,1 and its activity is stimulated by depletion of intracellular Ca2+ stores,1–4 receptor activation,5–6 and mechanical stretch.7 In VSMCs, TRPC1 is believed to be the main channel responsible for store-operated Ca2+ (SOC),1–2 a type of Ca2+ influx that is activated by depletion of intracellular Ca2+ stores.8 TRPC1 may coassemble with other TRP isoforms, including TRPC3, -P2, and -C4/-C5, to form heteromultimeric channels.1,4 TRPC1 may also interact with other proteins to form a signaling complex. For an example, TRPC1 interacts with a Ca2+-binding protein STIM1, and this interaction is crucial for TRPC1-mediated SOC.3
Functionally, TRPC1 activity is positively associated with VSMC proliferation1 and neointimal hyperplasia.9 However, there are conflicting reports regarding the role of TRPC1 in VSMC contraction.10–12 Some chronic treatments, such as hypoxia and organ cultures, were found to enhance TRPC1 protein expression with a parallel increase in vascular contractility.11–13 However, 2 other studies argued against any linkage between SOC and an increased vascular contractility in freshly isolated arteries.10,14 In yet another study, blockage of TRPC1 activity by an anti-TRPC1 antibody T1E3 was found to reduce endothelin-1-induced contraction in rat basilar artery but not in rat caudal artery.15
TRP channels allow influx of positive ions such as Na+ and Ca2+. Such a cation influx is expected to result in membrane depolarization.16 Indeed, it has been shown that activity of TRPC3, -C6, and -M4 results in membrane depolarization,17 and this depolarization subsequently activates voltage-gated Ca2+ channels in VSMCs, leading to vascular contraction.17 Interestingly, a recent study found that activation of another TRP channel, TRPV4, results in membrane hyperpolarization. In this instance, TRPV4 forms a Ca2+ signaling complex with ryanodine receptor and BKCa. Ca2+ influx through TRPV4 channel preferentially stimulates ryanodine receptor in the sarcoplasmic reticulum, generating Ca2+ sparks that signal adjacent BKCa to open and cause membrane hyperpolarization.18 Such a coupling between BKCa-TRPV4-ryanodine receptor is believed to be the underlying mechanism for epoxyeicosatrienoic acid-mediated endothelium-derived hyperpolarizing factor activity in some vascular beds.18
In the present study, we investigated possible interaction between TRPC1 and BKCa in VSMCs. Our results show that TRPC1 physically associates with BKCa to form a signaling complex and that Ca2+ influx through TRPC1 activates BKCa to induce membrane hyperpolarization in VSMCs. This hyperpolarizing effect of TRPC1-BKCa coupling may serve to prevent excessive contraction of VSMCs to contractile agonists.
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Materials and Methods |
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Cell Culture
All animal experiments were conducted in accordance with NIH publication no. 8523. Primary cultured VSMCs were isolated from male Sprague-Dawley rats. Briefly, thoracic aorta was dissected. After rubbing off endothelial layer, smooth muscle layers were peeled off and then digested with 0.2% collagenase type 1A and 0.9% papain for 1 hour. The dispersed VSMCs were cultured for 5 to 7 days before experimental use. VSMCs and HEK293 cells were both cultured in DMEM supplemented with 10% FBS.
[Ca2+]i Measurement
Cytosolic Ca2+ ([Ca2+]i) was measured as described elsewhere.19 Briefly, cells were loaded with 10 µmol/L Fluo-3/AM. Ca2+ stores was depleted by treating VSMCs with 4 µmol/L thapsigargin for 6 to 8 minutes in a Ca2+-free physiological saline (0Ca2+-PSS), which contained (in mmol/L): 140 NaCl, 5 KCl, 1 MgCl2, 10 glucose, 0.2 EGTA, 5 Hepes, pH 7.4. Ca2+ influx was initiated by applying 1 mmol/L extracellular Ca2+. The cells were pretreated with/without T1E3 (1:200) or antigen-preabsorbed T1E3 for 1 hour before experiments. Fluorescence signal was recorded by Fluoview FV1000 confocal laser scanning system. Changes in [Ca2+]i were displayed as a ratio of fluorescence relative to the intensity before the application of extracellular Ca2+ (F1/F0).
Membrane Potential Measurement
For membrane potential measurement using potentiometric fluorescence dye bis-oxonol [DiBAC4(3)], primary cultured VSMCs or endothelium-denuded aortic strips (3 mm wide by 5 mm long) were loaded with 100 nmol/L DiBAC4(3) at 37°C for 10 minutes. The tissues or cells were treated with/without iberiotoxin (50 nmol/L) at 37°C for 10 minutes or with/without T1E3 (1:200) or antigen-preabsorbed T1E3 overnight at 4°C for tissues or 1 hour at room temperature for cultured cells. Change in fluorescence was measured by FV1000 confocal system. A quantitative relationship between changes in DiBAC4(3) fluorescence versus membrane potential was established using Na+ ionophore gramicidin in Na+-free media.20
The methods for sharp microelectrode measurement was as described elsewhere.21 Briefly, segments of mesenteric arteries was cut open. After rubbing off endothelial layer, a conventional sharp microelectrode filled with 3 mol/L KCl (tip resistance 40 to 80 M
) was inserted into smooth muscle cells from the lumen side. The artery segments were preincubated with T1E3 (1:50) or preimmune IgG (1:50) at 4°C overnight or with/without iberiotoxin (50 nmol/L) at 37°C for 10 minutes.
Arterial Tension Measurement
Segments of the secondary and tertiary branches of rat mesenteric artery (
2 to 3 mm long) were dissected, and endothelial layer was rubbed off. The segments were mounted in a DMT myograph (model 610M) under a normalized tension as previously described.22 Contractile agonists were added in a cumulative fashion to the bath to obtain concentration-response curves. The contractions were expressed as active wall tension (WT)=F/2x, where F stands for the force in millinewtons and x for the longitudinal length of the vessels in millimeters. The artery segments were preincubated with T1E3 (1:50), antigen-preabsorbed T1E3, or preimmune IgG (1:50) at 4°C overnight. Iberiotoxin incubation (50 nmol/L) was at 37°C for 10 minutes. T1E3 and preimmune IgG were applied in similar quantities to balance possible osmotic effect.
Preparation of T1E3 and Preimmune IgG
T1E3 antibody was raised in rabbits using the strategy developed by Xu et al23 Briefly, a peptide corresponding to TRPC1 putative pore region (CVGIFCEQQSNDTFHSFIGT) was synthesized and conjugated to keyhole limpet hemocyanin (KLH) at Alpha Diagnostic International. The coupled T1E3 peptide was injected into the tail vein of a rabbit followed by two boost doses. T1E3 antiserum was collected 4 weeks after the second boost. IgG was purified from T1E3 antiserum and preimmune serum using a protein G column.
In antigen preabsorption control, T1E3 was preabsorbed with excessive amount of peptides (1:16 weight ratio) for 2.5 hours at room temperature.
Cloning and Transfection
Human BKCa cDNA (NM_002247) was a gift from Dr Desir GV (Yale University). Human TRPC1 cDNA (NM_003304) was obtained by RT-PCR from human coronary endothelial cells. Both genes were cloned into pcDNA6 vector.
Transfection condition was as described elsewhere.24 Briefly, HEK293 cells were transfected with constructs containing TRPC1 and/or BKCa using Lipofectamine 2000 in 6-well plates. Approximately 80% of HEK293 cells were successfully transfected as determined by a control transfection using a GFP-expressing pcDNA6. Functional studies were performed 3 days posttransfection.
Immunoprecipitation and Immunoblots
Immunoprecipitation and immunoblots were as described elsewhere.24 To prepare smooth muscle cell lysates, smooth muscle layers were obtained by peeling off from the adventitial layers with forceps, followed by homogenization. The proteins were extracted from the lysates of smooth muscle cells or HEK293 cells with detergent extracted buffer, which contained 1% Nonidet P-40, 150 mmol/L NaCl, 20 mmol/L Tris-HCl, pH 8.0, with addition of protease inhibitor cocktail tablets. Extracted proteins (800 µg) were then incubated with 7 µg of anti-TRPC1 (Alomone Laboratory) or anti-BKCa (Alomone Laboratory) on a rocking platform overnight at 4°C. Protein A agarose was then added, followed by a further incubation at 4°C for 3 hours. The immunoprecipitates were washed and resolved on an 8% SDS-PAGE gel.
For immunoblots, the poly(vinylidene difluoride) membrane carrying transferred proteins was incubated at 4°C overnight with the primary anti-TRPC1 (1:200), anti-BKCa (1:200), or T1E3 (1:200). Immunodetection was accomplished using horseradish peroxidase-conjugated secondary antibody, followed by ECL detection system.
Double-Labeling Immunofluorescence Assay
Double immunofluorescence assay was performed as described elsewhere.25 Briefly, freshly dispersed rat aortic VSMCs were fixed with 3.7% formaldehyde and permeabilized with 0.1% Triton X-100. Nonspecific immunostaining was blocked by preincubating the cells with 2% BSA. The cells were incubated with a mixture of T1E3 (1:50, raised in rabbit) and anti-BKCa (1:50, raised in goat) at 4°C overnight, followed by incubation with a mixture of secondary goat anti-rabbit IgG-conjugated to Alexa Fluor 488 (1:200) and rabbit anti-goat IgG-conjugated to Alexa Fluor 546 (1:100). Immunofluorescence was detected by FV1000 confocal system. A quantitative analysis of the colocalization was carried out through pixel-by-pixel correlation of red and green images using FV10-ASW 1.5 software.
An expanded Materials and Methods section is available in the online data supplement at http://circres.ahajournals.org.
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Results |
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SOC and Its Associated Membrane Hyperpolarization in Primary Cultured VSMCs
We first studied SOC in primary cultured rat aortic VSMCs. The cells were treated with thapsigargin (4 µmol/L) for 6 to 8 minutes in 0Ca2+-PSS to deplete intracellular Ca2+ stores, which resulted in a rise in [Ca2+]i (Figure 1A). Subsequent application of extracellular Ca2+ (1 mmol/L) elicited the SOC (Figure 1A and 1E). In control cells without thapsigargin treatment, application of extracellular Ca2+ had no effect on [Ca2+]i level (Figure 1B and 1E).
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The role of SOC in modulating membrane potential was examined using a potentiometric fluorescence dye DiBAC4(3).26 Initiation of SOC by applying extracellular Ca2+ caused a marked membrane hyperpolarization in VSMCs as indicated by 21±3% (n=8) decrease in DiBAC4(3) fluorescence (Figure 1C and 1F). In control cells without store depletion, addition of extracellular Ca2+ had no effect on membrane potential (Figure 1D and 1F). These data suggest that SOC induces membrane hyperpolarization in VSMCs.
To calibrate the changes of DiBAC4(3) fluorescence versus the membrane potential, a standard curve was established (Figure I in the online data supplement). Based on this standard curve, a 21% decrease in DiBAC4(3) fluorescence was equivalent to a hyperpolarization of 24 mV (n=14). Whole cell patch clamp was used to verify this hyperpolarization. Under the same conditions as in DiBAC4(3) experiments, SOC-induced membrane hyperpolarization measured by whole cell patch clamp was 28±4 mV (n=14) (supplemental Figure II), which was close to the value estimated by DiBAC4(3) method.
Role of TRPC1 in SOC and Its Associated Membrane Hyperpolarization in Primary Cultured VSMCs
We used a polyclonal antibody T1E3 that can plug the pore of TRPC1.2 Preincubation of cells with T1E3 (1:200) for 1 hour completely abolished SOC (Figure 2A, 2B, and 2H). This blocking effect was absent after T1E3 was preabsorbed by excessive amount of peptide antigen (Figure 2C and 2H), confirming that the blockage was attributable to the specific action of T1E3 on TRPC1. These data agreed well with the results from other labs,1 supporting the notion that TRPC1 is the main component of SOC in VSMCs.
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Effect of T1E3 on the SOC-induced membrane hyperpolarization was then tested. Incubation of VSMCs with T1E3 (1:200) for 1 hour diminished this hyperpolarization (Figure 2D, 2F, and 2I). For controls, incubation with antigen-preabsorbed T1E3 or bovine serum albumin (BSA) for 1 hour had no effect on the hyperpolarization (Figure 2E, 2G, and 2I). Furthermore, verapamil (10 µmol/L) had no effect on this hyperpolarization, indicating that voltage-gated Ca2+ channels were not involved (supplemental Figure III).
Role of BKCa in SOC-Induced Membrane Hyperpolarization of Primary Cultured VSMCs
Presumably, cation influx through TRPC1 should result in membrane depolarization instead of hyperpolarization. Therefore, we hypothesized that Ca2+ influx through TRPC1 may activate BKCa, resulting in membrane hyperpolarization. Two selective BKCa blockers, iberiotoxin and charybdotoxin, were used to test this hypothesis. Both agents at 50 nmol/L completely abolished the SOC-induced membrane hyperpolarization (Figure 3A through 3D), supporting a functional association of TRPC1 with BKCa.
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In the present study, T1E3 was used as the main tool to determine the functional involvement of TRPC1. Therefore, we needed to exclude the possibility of a direct T1E3 action on BKCa activity. In whole-cell patch clamp experiments, we recorded a large iberiotoxin-sensitive current in VSMCs, which could be attributed to BKCa (supplemental Figure IV). T1E3 had no effect on this current, indicating that T1E3 did not directly inhibit BKCa activity (supplemental Figure IV). Another concern is whether TRPC1 is coupled to intermediate conductance Ca2+-sensitive K+ channel (IKCa), the expression of which was reported to be upregulated during VSMC culture.27 Presumably, such a coupling could also contribute to the SOC-induced membrane hyperpolarization. In experiments, a low level of IKCa expression was indeed detected in the primary cultured VSMCs (supplemental Figure V). However, inhibition of IKCa activity with TRAM-34 (10 µmol/L) had no effect on the SOC-induced membrane hyperpolarization (Figure 3D), suggesting that IKCa was not involved.
Role of BKCa and TRPC1 in SOC-Induced Membrane Hyperpolarization of VSMCs in Intact Vascular Tissues
Primary cultured VSMCs may not fully represent the cells in vivo, because cell isolation and culture procedures could cause phenotypic drift. Therefore, intact vascular tissues were used to verify the above findings. In one series of experiments, endothelium-denuded aortic strips were loaded with DiBAC4(3). The strips bathed in 0Ca2+-PSS were treated with thapsigargin (4 µmol/L, 20 minutes) to deplete Ca2+ stores. Subsequent application of extracellular Ca2+ (1 mmol/L) induced membrane hyperpolarization in VSMCs within the strips (Figure 4A and 4E). The hyperpolarization was inhibited by iberiotoxin (50 nmol/L, 10 minutes) (Figure 4D and 4E) and T1E3 (1:200, overnight) (Figure 4B and 4E). Antigen-preabsorbed T1E3 had no blocking effect (Figure 4C and 4E). Similar results were also obtained by sharp microelectrode methods, in which the membrane potential of VSMCs in intact rat mesenteric arteries was measured directly (Figure 5A through 5C). Taken together, these data strongly suggest that TRPC1 is indeed functionally coupled to BKCa in VSMCs in vivo.
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Note that, in the absence of extracellular Ca2+, application of thapsigargin (4 µmol/L) caused a small depolarization in VSMCs (Figure 1C and 5
A). The reason for this depolarization was unclear, but it could result from Na+ influx through store-operated cation channels, mainly TRPC1. In experiments, T1E3 treatment reduced this depolarization (Figure 5B and 5C), supporting the involvement of TRPC1 in this depolarization.
Effect of T1E3 and Iberiotoxin on Phenylephrine-, Endothelin-1–, and U46619-Induced Membrane Depolarization and Contraction in Rat Mesenteric Arteries
We then tested whether TRPC1-BKCa coupling has any role in modulating agonist-induced membrane depolarization and vascular contraction. Several physiologically relevant agents, including an 1-adrenoceptor agonist phenylephrine, endothelin-1, and a thromboxane mimetic, U46619, were used. Sharp microelectrode and isometric tension studies showed that application of these agonists caused dose-dependent membrane depolarization (Figure 5D through 5F) and contraction (Figure 6) of VSMCs in isolated rat mesenteric arteries. Importantly, a preincubation of the vessels with T1E3 (1:50, overnight) or iberiotoxin (50 nmol/L, 10 minutes) enhanced the depolarization (Figure 5D through 5F) and contraction (Figure 6) to all 3 agonists. Antigen-preabsorbed T1E3 had no effect on the contraction (Figure 6). These data support a role of TRPC1-BKCa in agonist-induced membrane depolarization and vascular contraction.
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Note that in the presence of iberiotoxin, T1E3 had no additional effect on agonist-induced vascular contraction (supplemental Figure VI). This is consistent with the notion that TRPC1 exerts its effect through BKCa. However, it appeared that iberiotoxin was more effective than T1E3 in enhancing the agonist-induced depolarization (Figure 5D through 5F) and contraction (Figure 6). This could either be attributable to a poor permeability of T1E3 in vascular tissues or attributable to existence of an alternative TRPC1-independent BKCa activation pathway.
Physical Association of TRPC1 With BKCa()
The above results indicate that TRPC1 and BKCa are functionally linked. We next used coimmunoprecipitation method to determine whether these two proteins are physically associated. Two antibodies used for coimmunoprecipitation, anti-TRPC1 and anti-BKCa() (both from Alomone Laboratory), were previously reported to be highly specific to their targets.7,28 Our immunoblot experiments confirmed that 2 antibodies recognized a single band in TRPC1- and BKCa-transfected HEK cells respectively (Figure 7A and 7D). The specificity of T1E3 was also verified by immunoblots, in which T1E3 recognized the expected band whereas antigen-preabsorbed T1E3 had no band (Figure 7A). Importantly, coimmunoprecipitation experiments demonstrated that anti-BKCa() antibody was able to pull down TRPC1 in the proteins lysates freshly prepared from rat aortic smooth muscle layers (Figure 7B). Furthermore, anti-TRPC1 antibody was able to reciprocally pull down BKCa() (Figure 7C). In control experiments [labeled as IP(-) in Figure 7B and 7C], the pull-down experiments were performed using preimmune IgG. As expected, no band could be observed [IP(-) in Figure 7B and 7C]. Taken together, these data suggest that TRPC1 physically associates with BKCa(), forming a signaling complex.
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To further confirm that TRPC1 and BKCa() indeed have physical interaction, we next used the HEK293 cells that overexpress both TRPC1 and BKCa. The results showed that anti-BKCa() antibody could pull down TRPC1, and anti-TRPC1 antibody could reciprocally pull down BKCa() (supplemental Figure VII).
Colocalization of TRPC1 and BKCa() at Subcellular Level
Double-labeling immunofluorescence experiments were performed to determine subcellular localization of TRPC1 and BKCa() in freshly dispersed rat aortic VSMCs. Cells were stained for TRPC1 with Alexa fluor 488 (green) and for BKCa() with Alexa fluor 546 (red). As shown in Figure 8, both TRPC1 and BKCa() were mostly localized on the plasma membrane. On merged images, there was very strong overlapping of TRPC1 and BKCa() fluorescence (yellow) (Figure 8C). Quantitative colocalization analysis showed that 88±1% (n=38) of TRPC1 labeling was colocalized with BKCa(), and conversely 85±2% (n=38) of BKCa() labeling was colocalized with TRPC1 (Figure 8G). In control experiments, there was no staining if the primary antibodies were preabsorbed with excessive amounts of respective antigens (Figure 8E and 8F). These data suggest that TRPC1 is colocalized with BKCa() in rat aortic VSMCs.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The major findings of this study are as follows: (1) in the primary cultured VSMCs, SOC was mediated by TRPC1, and furthermore this Ca2+ influx induced membrane hyperpolarization; (2) this hyperpolarization was diminished in the presence of BKCa channel blockers or an anti-TRPC1 blocking antibody T1E3; (3) the above functional results were confirmed in VSMCs of intact arteries using both potentiometric fluorescence dye and sharp microelectrode methods; (4) Inhibition of TRPC1 activity by T1E3 markedly enhanced the membrane depolarization and contraction of VSMCs in response to several physiologically-relevant contractile agonists; (5) coimmunoprecipitation experiments showed that an anti-BKCa(
) antibody could pull down TRPC1, and furthermore an anti-TRPC1 antibody could reciprocally pull down BKCa(); and (6) double-labeling immunocytochemistry showed that TRPC1 and BKCa were colocalized in the same subcellular regions, mainly on the plasma membrane, in VSMCs. Taken together, these data suggest that TRPC1 and BKCa are physically associated with each other in VSMCs, and that Ca2+ influx through TRPC1 activates BKCa, leading to membrane hyperpolarization. The hyperpolarizing effect of TRPC1-BKCa coupling could serve to reduce agonist-induced membrane depolarization, thereby preventing excessive contraction of VSMCs to contractile agonists.
Mounting evidence suggests that TRPC1 associates with other proteins to form a signaling complex that can include inositol trisphosphate receptor, homer, calmodulin, caveolin-1, and myxovirus-resistance protein A.1 In the present study, we found that TRPC1 physically associates with BKCa(), and this interaction links TRPC1 to membrane hyperpolarization of VSMCs. These results add a new dimension to the role of TRPC1 channel. The physical coupling between BKCa and TRPC1 would allow an efficient signal transduction between TRPC1 and BKCa. Furthermore, because TRPC1 coassembles with other TRP channels to form heterotetramers in vivo, our model also allows an interaction of BKCa with other assembly partners of TRPC1, including TRPC4, -C5, and -P2. The presence of such a signaling complex, which contains BKCa, TRPC1 and its assembly partners, would allow diverse signals, including store-depletion, receptor activation, hypoosmotic cell swelling and oxidative stress, to converge on this signaling complex to activate BKCa, thereafter inducing hyperpolarization in VSMCs.1,29
Both BKCa and TRPC1 are abundantly expressed in almost all types of smooth muscle cells.1,30 We expect that the membrane hyperpolarization caused by TRPC1-BKCa coupling would inactivate voltage-gated Ca2+ channels, which are the dominant Ca2+ influx channels in VSMCs, thus serving to reduce vascular tone. We subsequently tested this hypothesis using rat mesenteric arteries. Several physiologically relevant agents, including phenylephrine, endothelin-1, and U46619, were used to depolarize VSMCs and to contract the vessels. It is well documented that these contractile agents induce vascular contraction at least partly by stimulating Ca2+ release from intracellular Ca2+ stores.31–33 These agonists also cause membrane depolarization,31,34 which would increase Ca2+ influx through voltage-gated Ca2+ channels. In addition, the store Ca2+ release is expected to result in an enhanced SOC, which is known to be mediated by TRPC1 in VSMCs.1 Based on our present model of TRPC1-BKCa coupling, we reasoned that this enhanced SOC would tend to have a hyperpolarizing effect and might thus reduce agonist-induced membrane depolarization and contraction in VSMCs. Our prediction was indeed confirmed. Sharp microelectrode study showed that blockage of TRPC1 by T1E3 antibody caused a marked increase in membrane depolarization in response to phenylephrine, endothelin-1, and U46619. Isometric tone studies showed that T1E3 enhanced vascular contraction in response to these three agonists. These results suggest that TRPC1-BKCa coupling has an important functional role in modulating agonist-induced vascular contraction.
Our results appear to conflict with a previous report by Bergdahl et al,15 who demonstrated that T1E3 treatment reduced endothelin-1-induced contraction in rat basilar artery but not in caudal artery. The reason for this discrepancy is not clear. One possibility is the variations in vessel types used in two studies. TRPC1 and/or BKCa could be expressed at different level in different vascular beds, and their coupling could also vary. These may contribute to the altered vascular responses. Several previous studies also showed that some chronic treatments such as hypoxia and organ cultures enhanced TRPC1 protein expression in arteries with a parallel increase in vascular contractility.11–13 However, these data are not in direct conflict with our model, because the overexpressed TRPC1 under chronic treatments might not be physically coupled to BKCa. Without TRPC1-BKCa coupling, Ca2+ influx through overexpressed TRPC1 is indeed expected to cause vascular contraction instead of relaxation.
The physiological roles of TRPC1-BKCa coupling may not be limited to vascular tone control. TRPC1 and BKCa channels are expressed across many cell types,1,35 and both are suggested to play a key role in a variety of other body function including VSMC proliferation,36 neointimal hyperplasia,9 neurotransmission,1,35 neuronal growth cone formation,1,37 and cell volume regulation.1,38 Thus, it is possible that TRPC1-BKCa signal complex may play a more general role in other body function. Consistent with this notion, our coimmunoprecipitation data showed that, when overexpressed in HEK293 cells, BKCa and TRPC1 also bind to each other to form a protein complex (supplemental Figure VII). Furthermore, a physical coupling between TRPC1 and BKCa was also found in vascular endothelial cells (data not shown).
In conclusion, we demonstrated that TRPC1 and BKCa are physically associated with each other. Ca2+ influx through TRPC1 activates BKCa, leading to membrane hyperpolarization in VSMCs. This hyperpolarizing effect of TRPC1-BKCa coupling could serve to prevent excessive contraction of VSMCs to contractile agonists.
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Acknowledgments |
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Sources of Funding
This study was supported by Hong Kong Research Grants Council grants CUHK477307 and CUHK477408 and Li Ka Shing Institute of Health Sciences.
Disclosures
None.
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Footnotes |
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*Both authors contributed equally to this study.
Original received January 7, 2008; resubmission received October 2, 2008; revised resubmission received December 23, 2008; accepted January 13, 2009.
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References |
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